Systems and methods for determining cross-linking distribution in a cornea and/or structural characteristics of a cornea

ABSTRACT

In a corneal measurement system, an optical element focuses an excitation light to an area of corneal tissue at a selected depth. In response, a fluorescing agent applied to the cornea generates a fluorescence emission. An aperture of a pinhole structure selectively transmits the fluorescence emission from the area of corneal tissue at the selected depth. A detector captures the selected fluorescence emission transmitted by the aperture and communicates information relating to a measurement of the selected fluorescence emission captured by the detector. A controller receives the information from the detector and determines a measurement of the fluorescing agent in the area of corneal tissue at the selected depth. The system may include a scan mechanism that causes the optical element to scan the cornea at a plurality of depths, and the controller may determine a measurement of the fluorescing agent in the cornea as a function of depth.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application the benefit of, and priority to, U.S.Provisional Patent Application Ser. No. 62/444,910, filed on Jan. 11,2017, and U.S. Provisional Patent Application Ser. No. 62/573,440, filedon Oct. 17, 2017, the contents of these application being incorporatedentirely herein by reference.

BACKGROUND Field

The present disclosure pertains to systems and methods for eyetreatments or procedures, and more particularly, to systems and methodsthat determine a distribution of a cross-linking agent in a corneaand/or to determine structural characteristics of a cornea, e.g.,corneal thickness.

Description of Related Art

Cross-linking treatments may be employed to treat eyes suffering fromdisorders, such as keratoconus. In particular, keratoconus is adegenerative disorder of the eye in which structural changes within thecornea cause it to weaken and change to an abnormal conical shape.Cross-linking treatments can strengthen and stabilize areas weakened bykeratoconus and prevent undesired shape changes.

Cross-linking treatments may also be employed after surgical procedures,such as Laser-Assisted in situ Keratomileusis (LASIK) surgery. Forinstance, a complication known as post-LASIK ectasia may occur due tothe thinning and weakening of the cornea caused by LASIK surgery. Inpost-LASIK ectasia, the cornea experiences progressive steepening(bulging). Accordingly, cross-linking treatments can strengthen andstabilize the structure of the cornea after LASIK surgery and preventpost-LASIK ectasia.

Cross-linking treatments may also be employed to induce refractivechanges in the cornea to correct disorders such as myopia, hyperopia,myopia, hyperopia, astigmatism, irregular astigmatism, presbyopia, etc.

SUMMARY

According to aspects of the present disclosure, systems and methodsemploy illumination and imaging techniques to determine a distributionof a cross-linking agent in a cornea. Additionally or alternatively,systems and methods employ illumination and imaging techniques todetermine structural characteristics of a cornea, such as cornealthickness.

According to one embodiment, a measurement system for a cornea includesa light source configured to emit an excitation light that causes afluorescing agent, e.g., a cross-linking agent, applied to a cornea togenerate a fluorescence emission. The system includes an optical elementpositioned to receive the excitation light from the light source andconfigured to focus the excitation light to an area of corneal tissue ata selected depth of the cornea. The fluorescing agent in the corneagenerates the fluorescence emission in response to the excitation light.The system includes a pinhole structure including an aperture. Thepinhole structure is positioned to receive the fluorescence emissionfrom the fluorescing agent in the cornea. The aperture is configured toselectively transmit the fluorescence emission from the area of cornealtissue at the selected depth. The system includes a detector positionedto capture the selected fluorescence emission transmitted by theaperture and to communicate information relating to a measurement of theselected fluorescence emission captured by the detector. The systemincludes a controller communicatively coupled to the detector andconfigured to receive the information from the detector and todetermine, based on the information, a measurement of the fluorescingagent in the area of corneal tissue at the selected depth.

In some cases, the measurement system may include a scan mechanismconfigured to cause the optical element to scan the cornea at aplurality of depths and to focus the excitation light on a respectivearea of corneal tissue at each depth. For each depth: (i) the apertureof the pinhole structure is configured to selectively transmit thefluorescence emission from the respective area of corneal tissue, and(ii) the detector is configured to capture the selected fluorescenceemission transmitted by the aperture and configured to communicateinformation relating to a measurement of the selected fluorescenceemission captured by the detector. The controller is configured toreceive the information from the detector for each depth and todetermine, based on the information for the plurality of depths, ameasurement of the fluorescing agent in the cornea as a function ofdepth. The plurality of depths may extend from an anterior surface ofthe cornea to a posterior surface of the cornea, and the controller maybe further configured to determine, based on the information for theplurality of depths, at least one of a location of the posteriorsurface, a distance between the anterior surface and the posteriorsurface, or a location of an interface between sections of the cornea.

According to another embodiment, a measurement system for a corneaincludes a light source configured to emit an incidence light. Thesystem includes an optical element positioned to receive the incidencelight from the light source and configured to focus the incidence lightto an area of corneal tissue at a selected depth of the cornea. The areaof corneal tissue reflects the incidence light. The system includes ascan mechanism configured to cause the optical element to scan thecornea at a plurality of depths and to focus the excitation light on arespective area of corneal tissue at each depth. The plurality of depthsextending from an anterior surface of the cornea to a posterior surfaceof the cornea. The system includes a pinhole structure including anaperture. The pinhole structure is positioned to receive, for eachdepth, the reflected light from the respective area of corneal tissue.The aperture is configured, for each depth, to selectively transmit thereflected light from the respective area of corneal tissue. The systemincludes a detector positioned to capture the selected reflected lighttransmitted by the aperture for each depth and configured tocommunicate, for each depth, information relating to a measurement ofthe selected reflected light captured by the detector. The systemincludes a controller communicatively coupled to the detector andconfigured to receive the information from the detector for each depthand to determine, based on the information for the plurality of depths,at least one of a location of the posterior surface, a distance betweenthe anterior surface and the posterior surface, or a location of asub-corneal interface.

According to yet another embodiment, a measurement system for a cornea,includes a light source configured to emit an excitation light thatcauses a fluorescing agent applied to a cornea to generate afluorescence emission. The system includes at least one optical elementpositioned to receive the excitation light from the light source andconfigured to deliver the excitation light. The excitation light extendsthrough a plurality of depths of the cornea. The fluorescing agent inthe cornea generates the fluorescence emission in response to theexcitation light. The system includes a detector positioned to capturean image of the fluorescence emission from the cornea. The systemincludes a controller communicatively coupled to the detector andconfigured to: receive the image from the detector, scan the image tomeasure the fluorescence emission at the plurality of depths, and todetermine a measurement of the fluorescing agent in the cornea as afunction of depth based on the measurement of the fluorescence emissionat the plurality of depths. The excitation light may further extend inat least one lateral direction as it extends through the plurality ofdepths of the cornea, and the controller may be further configured toscan the image to measure the fluorescence emission along the at leastone lateral direction at the plurality of depths, and to determine ameasurement of the fluorescing agent in the cornea as a function ofdepth and lateral location based on the measurement of the fluorescenceemission along the at least one transverse direction at the plurality ofdepths.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example system that delivers a cross-linking agentand photoactivating light to a cornea of an eye in order to generatecross-linking of corneal collagen, according to aspects of the presentdisclosure.

FIG. 2A illustrates a diagram for photochemical kinetic reactionsinvolving riboflavin and photoactivating light (e.g., ultraviolet A(UVA) light) applied during a corneal cross-linking treatment, accordingto aspects of the present disclosure.

FIG. 2B illustrates a diagram for parameters that can affect thephotochemical kinetic reactions shown in FIG. 2A.

FIG. 3 illustrates an example system for measuring fluorescenceassociated with a distribution of a photosensitizer, e.g., riboflavin,in an eye according to aspects of the present disclosure.

FIG. 4 illustrates another example system for measuring fluorescenceassociated with a distribution of a photosensitizer, e.g., riboflavin,in an eye according to aspects of the present disclosure.

FIG. 5 illustrates yet another example system for measuring fluorescenceassociated with a distribution of a photosensitizer, e.g., riboflavin,in an eye according to aspects of the present disclosure.

FIG. 6 illustrates a further example system for measuring fluorescenceassociated with a distribution of a photosensitizer, e.g., riboflavin,in an eye according to aspects of the present disclosure.

FIG. 7 illustrates yet a further example system for measuringfluorescence associated with a distribution of a photosensitizer, e.g.,riboflavin, in an eye according to aspects of the present disclosure.

FIG. 8 illustrates an additional example system for measuringfluorescence associated with a distribution of a photosensitizer, e.g.,riboflavin, in an eye according to aspects of the present disclosure.

FIG. 9 illustrates an example approach for measuring corneal thicknessaccording to aspects of the present disclosure.

While the present disclosure is susceptible to various modifications andalternative forms, a specific embodiment thereof has been shown by wayof example in the drawings and will herein be described in detail. Itshould be understood, however, that it is not intended to limit thepresent disclosure to the particular forms disclosed, but on thecontrary, the intention is to cover all modifications, equivalents, andalternatives falling within the spirit of the present disclosure.

DESCRIPTION

FIG. 1 illustrates an example treatment system 100 for generatingcross-linking of collagen in a cornea 2 of an eye 1. The treatmentsystem 100 includes an applicator 132 for applying a cross-linking agent130 to the cornea 2. In example embodiments, the applicator 132 may bean eye dropper, syringe, or the like that applies the photosensitizer130 as drops to the cornea 2. Example systems and methods for applyingthe cross-linking agent is described in U.S. patent application Ser. No.15/486,778, filed Apr. 13, 2017 and titled “Systems and Methods forDelivering Drugs to an Eye,” the contents of which are incorporatedentirely herein by reference.

The cross-linking agent 130 may be provided in a formulation that allowsthe cross-linking agent 130 to pass through the corneal epithelium 2 aand to underlying regions in the corneal stroma 2 b. Alternatively, thecorneal epithelium 2 a may be removed or otherwise incised to allow thecross-linking agent 130 to be applied more directly to the underlyingtissue.

The treatment system 100 includes an illumination system with a lightsource 110 and optical elements 112 for directing light to the cornea 2.The light causes photoactivation of the cross-linking agent 130 togenerate cross-linking activity in the cornea 2. For example, thecross-linking agent may include riboflavin and the photoactivating lightmay include ultraviolet A (UVA) (e.g., approximately 365 nm) light.Alternatively, the photoactivating light may include another wavelength,such as a visible wavelength (e.g., approximately 452 nm). As describedfurther below, corneal cross-linking improves corneal strength bycreating chemical bonds within the corneal tissue according to a systemof photochemical kinetic reactions. For instance, riboflavin and thephotoactivating light may be applied to stabilize and/or strengthencorneal tissue to address diseases such as keratoconus or post-LASIKectasia.

The treatment system 100 includes one or more controllers 120 thatcontrol aspects of the system 100, including the light source 110 and/orthe optical elements 112. In an implementation, the cornea 2 can be morebroadly treated with the cross-linking agent 130 (e.g., with an eyedropper, syringe, etc.), and the photoactivating light from the lightsource 110 can be selectively directed to regions of the treated cornea2 according to a particular pattern.

The optical elements 112 may include one or more mirrors or lenses fordirecting and focusing the photoactivating light emitted by the lightsource 110 to a particular pattern on the cornea 2. The optical elements112 may further include filters for partially blocking wavelengths oflight emitted by the light source 110 and for selecting particularwavelengths of light to be directed to the cornea 2 for photoactivatingthe cross-linking agent 130. In addition, the optical elements 112 mayinclude one or more beam splitters for dividing a beam of light emittedby the light source 110, and may include one or more heat sinks forabsorbing light emitted by the light source 110. The optical elements112 may also accurately and precisely focus the photo-activating lightto particular focal planes within the cornea 2, e.g., at a particulardepths in the underlying region 2 b where cross-linking activity isdesired.

Moreover, specific regimes of the photoactivating light can be modulatedto achieve a desired degree of cross-linking in the selected regions ofthe cornea 2. The one or more controllers 120 may be used to control theoperation of the light source 110 and/or the optical elements 112 toprecisely deliver the photoactivating light according to any combinationof: wavelength, bandwidth, intensity, power, location, depth ofpenetration, and/or duration of treatment (the duration of the exposurecycle, the dark cycle, and the ratio of the exposure cycle to the darkcycle duration).

The parameters for photoactivation of the cross-linking agent 130 can beadjusted, for example, to reduce the amount of time required to achievethe desired cross-linking. In an example implementation, the time can bereduced from minutes to seconds. While some configurations may apply thephotoactivating light at an irradiance of 5 mW/cm², larger irradiance ofthe photoactivating light, e.g., multiples of 5 mW/cm², can be appliedto reduce the time required to achieve the desired cross-linking. Thetotal dose of energy absorbed in the cornea 2 can be described as aneffective dose, which is an amount of energy absorbed through an area ofthe corneal epithelium 2 a. For example the effective dose for a regionof the corneal surface 2A can be, for example, 5 J/cm², or as high as 20J/cm² or 30 J/cm². The effective dosedescribed can be delivered from asingle application of energy, or from repeated applications of energy.

The optical elements 112 of the treatment system 100 may include adigital micro-mirror device (DMD) to modulate the application ofphotoactivating light spatially and temporally. Using DMD technology,the photoactivating light from the light source 110 is projected in aprecise spatial pattern that is created by microscopically small mirrorslaid out in a matrix on a semiconductor chip. Each mirror represents oneor more pixels in the pattern of projected light. With the DMD one canperform topography guided cross-linking. The control of the DMDaccording to topography may employ several different spatial andtemporal irradiance and dose profiles. These spatial and temporal doseprofiles may be created using continuous wave illumination but may alsobe modulated via pulsed illumination by pulsing the illumination sourceunder varying frequency and duty cycle regimes. Alternatively, the DMDcan modulate different frequencies and duty cycles on a pixel by pixelbasis to give ultimate flexibility using continuous wave illumination.Or alternatively, both pulsed illumination and modulated DMD frequencyand duty cycle combinations may be combined. This allows for specificamounts of spatially determined corneal cross-linking. This spatiallydetermined cross-linking may be combined with dosimetry, interferometry,optical coherence tomography (OCT), corneal topography, etc., forpre-treatment planning and/or real-time monitoring and modulation ofcorneal cross-linking during treatment. Aspects of a dosimetry systemare described in further detail below. Additionally, pre-clinicalpatient information may be combined with finite element biomechanicalcomputer modeling to create patient specific pre-treatment plans.

To control aspects of the delivery of the photoactivating light,embodiments may also employ aspects of multiphoton excitationmicroscopy. In particular, rather than delivering a single photon of aparticular wavelength to the cornea 2, the treatment system 100 maydeliver multiple photons of longer wavelengths, i.e., lower energy, thatcombine to initiate the cross-linking. Advantageously, longerwavelengths are scattered within the cornea 2 to a lesser degree thanshorter wavelengths, which allows longer wavelengths of light topenetrate the cornea 2 more efficiently than light of shorterwavelengths. Shielding effects of incident irradiation at deeper depthswithin the cornea are also reduced over conventional short wavelengthillumination since the absorption of the light by the photosensitizer ismuch less at the longer wavelengths. This allows for enhanced controlover depth specific cross-linking. For example, in some embodiments, twophotons may be employed, where each photon carries approximately halfthe energy necessary to excite the molecules in the cross-linking agent130 to generate the photochemical kinetic reactions described furtherbelow. When a cross-linking agent molecule simultaneously absorbs bothphotons, it absorbs enough energy to release reactive radicals in thecorneal tissue. Embodiments may also utilize lower energy photons suchthat a cross-linking agent molecule must simultaneously absorb, forexample, three, four, or five, photons to release a reactive radical.The probability of the near-simultaneous absorption of multiple photonsis low, so a high flux of excitation photons may be required, and thehigh flux may be delivered through a femtosecond laser.

A large number of conditions and parameters affect the cross-linking ofcorneal collagen with the cross-linking agent 130. For example, theirradiance and the dose of photoactivating light affect the amount andthe rate of cross-linking.

When the cross-linking agent 130 is riboflavin in particular, the UVAlight may be applied continuously (continuous wave (CW)) or as pulsedlight, and this selection has an effect on the amount, the rate, and theextent of cross-linking. If the UVA light is applied as pulsed light,the duration of the exposure cycle, the dark cycle, and the ratio of theexposure cycle to the dark cycle duration have an effect on theresulting corneal stiffening. Pulsed light illumination can be used tocreate greater or lesser stiffening of corneal tissue than may beachieved with continuous wave illumination for the same amount or doseof energy delivered. Light pulses of suitable length and frequency maybe used to achieve more optimal chemical amplification. For pulsed lighttreatment, the on/off duty cycle may be between approximately 1000/1 toapproximately 1/1000; the irradiance may be between approximately 1mW/cm² to approximately 1000 mW/cm² average irradiance, and the pulserate may be between approximately 0.01 HZ to approximately 1000 Hz orbetween approximately 1000 Hz to approximately 100,000 Hz.

The treatment system 100 may generate pulsed light by employing a DMD,electronically turning the light source 110 on and off, and/or using amechanical or opto-electronic (e.g., Pockels cells) shutter ormechanical chopper or rotating aperture. Because of the pixel specificmodulation capabilities of the DMD and the subsequent stiffnessimpartment based on the modulated frequency, duty cycle, irradiance anddose delivered to the cornea, complex biomechanical stiffness patternsmay be imparted to the cornea to allow for various amounts of refractivecorrection. These refractive corrections, for instance, may involvecombinations of myopia, hyperopia, astigmatism, irregular astigmatism,presbyopia and complex corneal refractive surface corrections because ofophthalmic conditions such as keratoconus, pellucid marginal disease,post-LASIK ectasia, and other conditions of corneal biomechanicalalteration/degeneration, etc. A specific advantage of the DMD system andmethod is that it allows for randomized asynchronous pulsed topographicpatterning, creating a non-periodic and uniformly appearing illuminationwhich eliminates the possibility for triggering photosensitive epilepticseizures or flicker vertigo for pulsed frequencies between 2 Hz and 84Hz.

Although example embodiments may employ stepwise on/off pulsed lightfunctions, it is understood that other functions for applying light tothe cornea may be employed to achieve similar effects. For example,light may be applied to the cornea according to a sinusoidal function,sawtooth function, or other complex functions or curves, or anycombination of functions or curves. Indeed, it is understood that thefunction may be substantially stepwise where there may be more gradualtransitions between on/off values. In addition, it is understood thatirradiance does not have to decrease down to a value of zero during theoff cycle, and may be above zero during the off cycle. Desired effectsmay be achieved by applying light to the cornea according to a curvevarying irradiance between two or more values.

Examples of systems and methods for delivering photoactivating light aredescribed, for example, in U.S. Patent Application Publication No.2011/0237999, filed Mar. 18, 2011 and titled “Systems and Methods forApplying and Monitoring Eye Therapy,” U.S. Patent ApplicationPublication No. 2012/0215155, filed Apr. 3, 2012 and titled “Systems andMethods for Applying and Monitoring Eye Therapy,” and U.S. PatentApplication Publication No. 2013/0245536, filed Mar. 15, 2013 and titled“Systems and Methods for Corneal Cross-Linking with Pulsed Light,” thecontents of these applications being incorporated entirely herein byreference.

The addition of oxygen also affects the amount of corneal stiffening. Inhuman tissue, O₂ content is very low compared to the atmosphere. Therate of cross-linking in the cornea, however, is related to theconcentration of O₂ when it is irradiated with photoactivating light.Therefore, it may be advantageous to increase or decrease theconcentration of O₂ actively during irradiation to control the rate ofcross-linking until a desired amount of cross-linking is achieved.Oxygen may be applied during the cross-linking treatments in a number ofdifferent ways. One approach involves supersaturating the riboflavinwith O₂. Thus, when the riboflavin is applied to the eye, a higherconcentration of O₂ is delivered directly into the cornea with theriboflavin and affects the reactions involving O₂ when the riboflavin isexposed to the photoactivating light. According to another approach, asteady state of O₂ (at a selected concentration) may be maintained atthe surface of the cornea to expose the cornea to a selected amount ofO₂ and cause O₂ to enter the cornea. As shown in FIG. 1, for instance,the treatment system 100 also includes an oxygen source 140 and anoxygen delivery device 142 that optionally delivers oxygen at a selectedconcentration to the cornea 2. Example systems and methods for applyingoxygen during cross-linking treatments are described, for example, inU.S. Pat. No. 8,574,277, filed Oct. 21, 2010 and titled “Eye Therapy,”U.S. Patent Application Publication No. 2013/0060187, filed Oct. 31,2012 and titled “Systems and Methods for Corneal Cross-Linking withPulsed Light,” the contents of these applications being incorporatedentirely herein by reference. Additionally, an example mask device fordelivering concentrations of oxygen as well as photoactivating light ineye treatments is described in U.S. Provisional Patent ApplicationPublication No. 2017/0156926, filed Dec. 3, 2016 and titled “Systems andMethods for Treating an Eye with a Mask Device,” the contents of whichare incorporated entirely herein by reference. For instance, a mask maybe placed over the eye(s) to produce a consistent and known oxygenconcentration above the surface.

When riboflavin absorbs radiant energy, especially light, it undergoesphotoactivation. There are two photochemical kinetic pathways forriboflavin photoactivation, Type I and Type II. Some of the reactionsinvolved in both the Type I and Type II mechanisms are as follows:

Common Reactions:

Rf→Rf₁*, l;   (r1)

Rf₁*→Rf, κ1;   (r2)

Rf₁*→Rf₃*, κ2;   (r3)

Type I reactions:

Rf₃*+DH→RfH.+D. κ3;   (r4)

2RfH.→Rf+RfH₂, κ4;   (r5)

Type II reactions:

Rf₃*+O₂→Rf+O1/2, κ5;   (r6)

DH+O1/2→D_(ox),   (r7)

D_(ox)+DH→D−D, κ7;   (r8)

In the reactions described herein, Rf represents riboflavin in theground state. Rf*₁ represents riboflavin in the excited singlet state.Rf*₃ represents riboflavin in a triplet excited state. Rf.⁻is thereduced radical anion form of riboflavin. RfH. is the radical form ofriboflavin. RfH₂ is the reduced form of riboflavin. DH is the substrate.DH.⁺is the intermediate radical cation. D. is the radical. D_(ox) is theoxidized form of the substrate.

Riboflavin is excited into its triplet excited state Rf*₃ as shown inreactions (r1) to (r3). From the triplet excited state Rf*₃, theriboflavin reacts further, generally according to Type I or Type IImechanisms. In the Type I mechanism, the substrate reacts with theexcited state riboflavin to generate radicals or radical ions,respectively, by hydrogen atoms or electron transfer. In Type IImechanism, the excited state riboflavin reacts with oxygen to formsinglet molecular oxygen. The singlet molecular oxygen then acts ontissue to produce additional cross-linked bonds.

Oxygen concentration in the cornea is modulated by UVA irradiance andtemperature and quickly decreases at the beginning of UVA exposure.Utilizing pulsed light of a specific duty cycle, frequency, andirradiance, input from both Type I and Type II photochemical kineticmechanisms can be employed to achieve a greater amount of photochemicalefficiency. Moreover, utilizing pulsed light allows regulating the rateof reactions involving riboflavin. The rate of reactions may either beincreased or decreased, as needed, by regulating, one of the parameterssuch as the irradiance, the dose, the on/off duty cycle, riboflavinconcentration, soak time, and others. Moreover, additional ingredientsthat affect the reaction and cross-linking rates may be added to thecornea.

If UVA radiation is stopped shortly after oxygen depletion, oxygenconcentrations start to increase (replenish). Excess oxygen may bedetrimental in the corneal cross-linking process because oxygen is ableto inhibit free radical photopolymerization reactions by interactingwith radical species to form chain-terminating peroxide molecules. Thepulse rate, irradiance, dose, and other parameters can be adjusted toachieve a more optimal oxygen regeneration rate. Calculating andadjusting the oxygen regeneration rate is another example of adjustingthe reaction parameters to achieve a desired amount of cornealstiffening.

Oxygen content may be depleted throughout the cornea, by variouschemical reactions, except for the very thin corneal layer where oxygendiffusion is able to keep up with the kinetics of the reactions. Thisdiffusion-controlled zone will gradually move deeper into the cornea asthe reaction ability of the substrate to uptake oxygen decreases.

Riboflavin is reduced (deactivated) reversibly or irreversibly and/orphoto-degraded to a greater extent as irradiance increases. Photonoptimization can be achieved by allowing reduced riboflavin to return toground state riboflavin in Type I reactions. The rate of return ofreduced riboflavin to ground state in Type I reactions is determined bya number of factors. These factors include, but are not limited to,on/off duty cycle of pulsed light treatment, pulse rate frequency,irradiance, and dose. Moreover, the riboflavin concentration, soak time,and addition of other agents, including oxidizers, affect the rate ofoxygen uptake. These and other parameters, including duty cycle, pulserate frequency, irradiance, and dose can be selected to achieve moreoptimal photon efficiency and make efficient use of both Type I as wellas Type II photochemical kinetic mechanisms for riboflavinphotosensitization. Moreover, these parameters can be selected in such away as to achieve a more optimal chemical amplification effect.

In addition to the photochemical kinetic reactions (r1)-(r8) above,however, the present inventors have identified the followingphotochemical kinetic reactions (r9)-(r26) that also occur duringriboflavin photoactivation:

Rf₃*→Rf, κ8;   (r9)

Rf₃*+Rf→2RfH., κ9;   (r10)

RfH₂+O₂→RfH.+H⁺+O₂ ⁻, κ10;   (r11)

RfH.+O₂→Rf+H⁺+O₂ ⁻, κ11:   (r12)

2Rf H₂+O₂ ⁻→2 RfH.+H₂O₂, κ12;   (r13)

2 RfH.+O₂ ⁻→2 Rf+H₂O₂, κ13;   (r14)

RfH.+H₂O₂→OH.+Rf+H₂O, κ14;   (r15)

OH.+DH→D.+H₂O, κ15;   (r16)

D.+D.→D−D, κ16; CXL   (r17)

O1/2→O₂, κ18;   (r18)

D.+RfH₂→RfH.+DH, κ19;   (r19)

$\begin{matrix}{{{Rf} + {{Rf}_{\underset{\kappa_{a}^{-}}{\leftarrow}}^{\overset{\kappa_{a}^{+}}{\rightarrow}}A_{1}}},\mspace{14mu} {\kappa_{a} = {\kappa_{a}^{+}/\kappa_{a}^{-}}}} & \left( {r\; 20} \right) \\{{{{RF}\; H_{2}} + {{Rf}\; {H_{2}}_{\underset{\kappa_{a}^{-}}{\leftarrow}}^{\overset{\kappa_{a}^{+}}{\rightarrow}}A_{2}}},\mspace{14mu} {\kappa_{a} = {\kappa_{a}^{+}/\kappa_{a}^{-}}}} & \left( {r\; 21} \right) \\{{{RF} + {{Rf}\; {H_{2}}_{\underset{\kappa_{b}^{-}}{\leftarrow}}^{\overset{\kappa_{b}^{+}}{\rightarrow}}A_{3}}},\mspace{14mu} {\kappa_{b} = {\kappa_{b}^{+}/\kappa_{b}^{-}}}} & ({r22})\end{matrix}$Rf₁*+A→Rf+A, κ_(1a)  (r23)

Rf₃*+A→Rf+A. κ_(3A)  (r24)

2O₂ ⁻→O₂+H₂O₂, κ₁₂  (r25)

OH^(°)+CXL→inert products, κ_(OH)  (r26)

FIG. 2A illustrates a diagram for the photochemical kinetic reactionsprovided in reactions (r1) through (r26) above. The diagram summarizesphotochemical transformations of riboflavin (Rf) under UVAphotoactivating light and its interactions with various donors (DH) viaelectron transfer. As shown, cross-linking activity occurs: (A) throughthe presence of singlet oxygen in reactions (r6) through (r8) (Type IImechanism); (B) without using oxygen in reactions (r4) and (r17) (Type Imechanism); and (C) through the presence of peroxide (H₂O₂), superoxide(O₂), and hydroxyl radicals (. OH) in reactions (r13) through (r17).

As shown in FIG. 2A, the present inventors have also determined that thecross-linking activity is generated to a greater degree from reactionsinvolving peroxide, superoxide, and hydroxyl radicals. Cross-linkingactivity is generated to a lesser degree from reactions involvingsinglet oxygen and from non-oxygen reactions. Some models based on thereactions (r1)-(r26) can account for the level of cross-linking activitygenerated by the respective reactions. For instance, where singletoxygen plays a smaller role in generating cross-linking activity, modelsmay be simplified by treating the cross-linking activity resulting fromsinglet oxygen as a constant.

All the reactions start from Rf₃*as provided in reactions (r1)-(r3). Thequenching of Rf₃* occurs through chemical reaction with ground state Rfin reaction (r10), and through deactivation by the interaction withwater in reaction (r9).

As described above, excess oxygen may be detrimental in cornealcross-linking process. As shown in FIG. 2A, when the system becomesphoton-limited and oxygen-abundant, cross-links can be broken fromfurther reactions involving superoxide, peroxide, and hydroxyl radicals.Indeed, in some cases, excess oxygen may result in net destruction ofcross-links versus generation of cross-links.

As described above, a large variety of factors affect the rate of thecross-linking reaction and the amount of biomechanical stiffnessachieved due to cross-linking. A number of these factors areinterrelated, such that changing one factor may have an unexpectedeffect on another factor. However, a more comprehensive model forunderstanding the relationship between different factors forcross-linking treatment is provided by the photochemical kineticreactions (r1)-(r26) identified above. Accordingly, systems and methodscan adjust various parameters for cross-linking treatment according tothis photochemical kinetic cross-linking model, which provides a unifieddescription of oxygen dynamics and cross-linking activity. The model canbe employed to evaluate expected outcomes based on differentcombinations of treatment parameters and to identify the combination oftreatment parameters that provides the desired result. The parameters,for example, may include, but are not limited to: the concentration(s)and/or soak times of the applied cross-linking agent; the dose(s),wavelength(s), irradiance(s), duration(s), and/or on/off duty cycle(s)of the photoactivating light; the oxygenation conditions in the tissue;and/or presence of additional agents and solutions.

As shown in FIG. 2B, aspects of the system of reactions can be affectedby different parameters. For instance, the irradiance at whichphotoactivating light is delivered to the system affects the photonsavailable in the system to generate Rf₃*for subsequent reactions.Additionally, delivering greater oxygen into the system drives theoxygen-based reactions. Meanwhile, pulsing the photoactivating lightaffects the ability of the reduced riboflavin to return to ground stateriboflavin by allowing additional time for oxygen diffusion. Of course,other parameters can be varied to control the system of reactions.

Further aspects of the photochemical kinetic reactions provided inreactions (r1)-(r26) are described in U.S. Patent ApplicationPublication No. 2016/0310319, filed Apr. 27, 2016 and titled “Systemsand Methods for Cross-Linking Treatments of an Eye,” the contents ofwhich are incorporated entirely herein by reference.

When light of a particular wavelength is applied to a cross-linkingagent, such as riboflavin, the light can excite the cross-linking agentand cause the cross-linking agent to fluoresce. As such, an excitationlight can be employed to cause a cross-linking agent in corneal tissueto fluoresce and determine how the cross-linking agent is distributed inthe corneal tissue. When an image of the cornea is taken during theapplication of the excitation light, the intensity (magnitude) of thefluorescence, for instance, can be measured to determine the amount,i.e., dose, of cross-linking agent taken up by the corneal tissue. Usingthese principles, dosimetry systems can determine the presence anddistribution of the cross-linking agent in the cornea by capturing oneor more images of the fluorescence from the cross-linking agent as itresponds to the excitation light. Aspects of such systems are described,for instance, in U.S. Pat. No. 9,020,580, issued Apr. 28, 2015 andtitled “Systems and Methods for Monitoring Time Based Photo Active AgentDelivery or Photo Active Marker Presence,” and U.S. Patent ApplicationPublication No. 2016/0338588, filed May 23, 2016 and titled “Systems andMethods for Monitoring Cross-Linking Activity for Corneal Treatments,”the contents of these applications being incorporating entirely hereinby reference. In particular, U.S. Pat. No. 9,020,580 discloses anexample dosimetry system that employs a modified Scheimpflugconfiguration. Meanwhile, U.S. Patent Application Publication No.2016/0338588 discloses the use of hyperspectral imaging to analyzefluorescence.

Currently available cross-linking treatment systems do not indicatewhether sufficient riboflavin is present in the stroma prior toinitiating cross-linking treatment. This can lead to increasedprocedural variability and sub-optimal clinical results. Advantageously,aspects of the present disclosure address this problem by providing aquantitative, depth-resolved measurement of riboflavin concentration,which can be compared to a previously-defined target value known toprovide efficacious cross-linking activity.

According to aspects of the present disclosure, embodiments specificallyconfigure aspects of a confocal fluorescence microscope to measureriboflavin distribution in the corneal stroma as a function of depth. Assuch, embodiments provide an indication of whether sufficient riboflavinis present at a defined stromal depth to proceed with cornealcross-linking treatment. Embodiments may be integrated with across-linking treatment system as described herein or may be astandalone measurement system. Embodiments are also suitable formeasuring fluorescence induced by the photoactivating UV illuminationapplied during the cross-linking treatment, and can thus measure theprogress of cross-linking activity in real time.

According to aspects of the present invention, systems and methods canachieve one or more of the following:

-   -   1. Measure time evolution of depth profiles of riboflavin in        corneal stroma at selected sites to allow cross-linking        treatment to begin at more effective time(s).    -   2. Determine the three-dimensional (3D) distribution of        riboflavin in corneal stroma as a function of time.    -   3. Measure depth profiles of cross-link concentration at treated        sites for keeping the treatment procedure under control.    -   4. Reconstruct 3D distribution of cross-link concentration after        treatment.    -   5. Locate hazy areas in corneal stroma.

To accomplish the foregoing, systems and methods may employ aspects ofone or more of the following techniques:

-   -   1. Fluorescence microscopy.    -   2. Confocal microscopy.    -   3. Scheimpflug photography.    -   4. 3D reconstruction, image deconvolution, image registration,        and other image processing techniques.

Existing confocal fluorescence microscopes require fluidimmersion/contact objectives, are highly complex and expensive, and usehigh laser intensities, making them unsuitable for commercial use inliving human corneas. Advantageously, aspects of the present disclosureaddress this problem by simplifying the measurement system in a mannerthat is optimized to provide an indication of sufficient riboflavinpresence.

To demonstrate aspects of the present disclosure, FIG. 3 illustrates anexample system 300 to measure a concentration as a function of depth ofexogenous cross-linking agent, e.g., riboflavin, applied to a cornea 2.The system 300 includes a laser or LED light source 302 that emits lightto excite the cross-linking agent in the cornea 2. For instance, thelight source 302 may emit UV excitation light, e.g., with a wavelengthof 365 nm. Or the light source 302 may emit blue excitation light, e.g.,with a wavelength of 458 nm, where in some cases, the light source 302may employ a white light in combination with a blue light narrow bandfilter.

As shown in FIG. 3, the excitation light from the light source 302 isdirected through an aperture of a light source pinhole structure 304 anda collimating lens 306. In some cases, the light source 302 may be fibercoupled. The excitation light is then directed to a dichroic filter (ormirror) 308. Due to the wavelength of the excitation light, the dichroicfilter 308 reflects the excitation light toward a lens 310, such as anobjective lens or similar optical element. The lens 310 focuses theexcitation light to an area of the cornea 2 at a given depth along thez-axis. The transverse (x-y plane) resolution of the excitation lightcan be greater than 10 μm, such as approximately 10 μm to approximately200 μm. In contrast, typical confocal microscopes require a transverseresolution of less than 10 μm.

In response to the excitation light, the cross-linking agent in thecornea 2 fluoresces. For instance, riboflavin in the cornea 2 may emitgreen fluorescence. The fluorescence emission travels through the lens310 and to the dichroic filter 308. In contrast to reflecting light withthe wavelength of the excitation light, the dichroic filter 308 allowsthe light with the fluorescence wavelength to pass to a color filter312. The color filter 312 transmits the fluorescence emission to a lens314, e.g., a tube lens, while blocking residual excitation light and/orother light not having a wavelength of the fluorescence emission. Thelens 314 focuses the fluorescence emission to a detector 318 via anaperture of a detector pinhole 316. The detector pinhole structure 316prevents light originating from above or below the given corneal depthfrom reaching the detector 318. In other words, the detector pinholestructure 316 prevents out-of-focus light from reaching the detector318. The pinhole structure 316 may be configured to allow a lowerresolution along the z-axis of greater than 10 μm, such as approximately10 μm to approximately 100 μm. In contrast, conventional confocalmicroscopes require a resolution of less than 10 μm. In general, thesystem 300 may provide a working distance in air of greater than 10 mmand does not require fluid immersion or physical contact with the eye.

The detector 318 may be a photodiode, a photomultiplier tube, or acamera. The image plane of the detector 318 is parallel to the lensplane of the lens 314 as well as the area of excited corneal tissuealong the x-y plane. This configuration allows the area of the excitedcorneal tissue to be uniformly in focus at the detector 318.Correspondingly, the detector pinhole structure 316 is parallel to theimage plane of the detector 318.

The detector 318 is employed to quantify the amount, e.g., intensity, offluorescence emitted from the excited area of the cornea at the givendepth. The system 300 may be operated in steps to deliver excitationlight to various respective depths of the cornea 2 and to detect theamount of fluorescence from the cross-linking agent at each depth. Asshown in FIG. 3, the system 300 includes a scan mechanism 320 thatcauses the system 300 to scan the cornea 2 at various depths along thez-axis. For instance, the scan mechanism 320 may be an mechanical orelectromechanical device that moves or operates the lens 310 and/orother elements of the system 300 to adjust the delivery of theexcitation light to other depths. According to one implementation, thesystem 300 may first be operated to excite an area at the anteriorsurface 2 a of the cornea, and then subsequently operated to exciteareas at a series of depths under the anterior surface 2 a to at leastapproximately 200 μm into the stroma 2 b. The distance between twoconsecutive depths for delivery of the excitation light may range fromapproximately 10 μm to approximately 100 μm.

The amount of fluorescence detected at a given depth indicates theamount of cross-linking agent at that depth. Thus, the system 300 may beemployed to confirm whether a sufficient amount of cross-linking agentis present for the start of a cross-linking treatment. Prior toproceeding with the cross-linking treatment, an amount of cross-linkingagent may need to exceed a predefined threshold value at a predefineddepth or set of depths.

A time series signal from the detector 318 may be sampled at a highfrequency and synchronized with the scan mechanism 310 that changes thescan depth by moving one or more elements of the system 300. Theinformation from the detector 318 may be employed to reconstruct ariboflavin concentration curve along the z-axis. For instance, the timeseries signal from the detector 318 can be processed to calculate: (1)the location of the posterior surface 2 c of the cornea 2, (2) theriboflavin concentration as a function of depth into the cornea 2, (3)the anterior surface 2 a of the cornea, (4) the distance betweenposterior surface 2 c and the anterior surface 2 a, and (5) the locationof sub-corneal interfaces such as the epithelial-stroma interface.

The system 300 may include auxiliary optics, such as an imaging cameraand alignment lasers, to assist in aligning the system 300 to a desiredx, y, z position to initiate a scan. Additionally, several scans may beaveraged together in order to increase the signal-to-noise ratio.

Furthermore, data processing algorithms may be employed to detect anincomplete scan. For instance, when a scan captures fluorescenceemission from the anterior surface of the cornea 2, the information fromthe detector 318 indicates a sudden increase in fluorescence signalfollowed by a gradual decline in the fluorescence signal as the scanmoves deeper from the anterior surface 2 a into the stroma 2 b. If theinformation from the detector 318 does not indicate the sudden increasein fluorescence signal, the scan likely did not capture the anteriorsurface 2 a and may be considered incomplete.

The system 300 may provide other useful information to enhance aspectsof the cross-linking treatment. For instance, the system 300 may beemployed periodically or continuously to monitor the amount offluorescence emitted by the cross-linking agent as a cross-linkingtreatment progresses. In particular, repeated scans over time mayindicate when cross-linking activity has progressed to a desired stromaldepth. Additionally, the location of the posterior surface of the corneaas detected by the system 300 can be used to align the treatment planeof the cross-linking treatment system.

In general, embodiments include an illumination path and an imagingpath. The illumination path directs a point illumination to the cornea2, and the imaging path collects fluorescence emission resulting fromexcitation by the point illumination. Each illumination and imaging pathcan use an on-axis or an off-axis configuration. The system 300 shown inFIG. 3 is an on-axis optical system. The system 300 employs an on-axisillumination path 300 a, where the area of excited corneal tissue alongthe x-y plane is perpendicular to the illumination path 300 a for theexcitation light. The system 300 also employs an on-axis imaging path300 b, where the area of excited corneal tissue along the x-y plane isperpendicular to the imaging path for the fluorescence emission.

In embodiments where both the illumination path and the imaging path areon-axis, the illumination path and imaging path can be on eitherreflective side or transmission side of the dichroic mirror. As shown inFIG. 3, for instance, the illumination path 300 a is on the reflectiveside of the dichroic filter 308, while the imaging path 300 b is on thetransmission side of the dichroic filter 308. In particular, theexcitation light from the light source 302 is directed to the dichroicfilter 308, which reflects the excitation light, e.g., 90°, toward thecornea 2 based on the wavelength of the excitation light. The dichroicfilter 308 allows the fluorescence emission to pass toward the detector318 based on the wavelength of the fluorescence emission.

On the other hand, FIG. 4 illustrates an example system 400, which issimilar to the system 300, but includes an illumination path 400 adisposed on the transmission side of a dichroic mirror 408 and animaging path 400 b disposed on the reflective side of the dichroicmirror 408. In particular, the excitation light from the light source302 is directed to the dichroic filter 408, which allows the excitationlight to pass toward the cornea 2 based on the wavelength of theexcitation light. Meanwhile, the dichroic filter 408 reflects thefluorescence emission, e.g., 90°, toward the detector 318 based on thewavelength of the fluorescence emission.

In contrast to the example systems 300, 400 above, FIG. 5 illustrates anexample system 500 employing an on-axis illumination path 500 a and anoff-axis imaging path 500 b. A light source 502 emits light to excitethe cross-linking agent in the cornea 2. The excitation light from thelight source 502 is directed through an aperture of a light sourcepinhole structure 504 and to a lens 510, such as an objective lens orsimilar optical element. The lens 510 focuses the excitation light to anarea of the cornea 2 along the x-y plane at a given depth along thez-axis. As shown in FIG. 5, the area of excited corneal tissue along thex-y plane is perpendicular to the illumination path 500 a for theexcitation light. As such, the illumination path 500 b is considered tobe on-axis.

In response to the excitation light, the cross-linking agent in thecornea 2 fluoresces. The fluorescence emission travels through a lens514 and a color filter 512. The lens 514 focuses the fluorescenceemission onto a detector 518. The color filter 512 blocks residualexcitation light and/or other light not having a wavelength of thefluorescence emission. The fluorescence emission passes through anaperture of a detector pinhole structure 516 a (solid line) oralternatively 516 b (dashed line), which prevents light originating fromabove or below the given corneal depth from reaching the detector 518.The detector 518 may be a photodiode, a photomultiplier tube, or acamera. The detector 518 may be employed to quantify the amount, e.g.,intensity, of fluorescence emitted from the excited area of the corneaat the given depth.

The area of excited corneal tissue along the x-y plane is notperpendicular to the imaging path 500 b for the fluorescence emission.In other words, the imaging path 500 b extends from the x-y plane at thegiven depth at an angle that is not equal to 90°. As such, the imagingpath 500 b is considered to be off-axis.

According to one embodiment, the image plane of the detector 518 isparallel to the lens plane of the lens 514. As shown in FIG. 4, thedetector pinhole structure 516 a is correspondingly parallel to theimage plane and the lens plane, i.e., perpendicular to the imaging path500 b.

According to an alternative embodiment, the image plane of the detector518 is not parallel to the lens plane of the lens 514. Instead, thedetector 518 and the lens 514 are arranged so that the image plane andthe lens plane are in a Scheimpflug configuration. In thisconfiguration, the excited area of corneal tissue along the x-y plane ismore uniformly in focus at the detector 518, even though the imagingpath 500 b is not perpendicular to the excited area of corneal tissue.The detector pinhole structure 516 b is correspondingly angled relativeto the image plane and the lens plane, e.g., parallel to the x-y plane,to prevent light originating from above or below the given corneal depthfrom reaching the detector 518.

FIG. 6 illustrates an example system 600 employing an off-axisillumination path 600 a and an off-axis imaging path 600 b. A lightsource 602 emits a light to excite the cross-linking agent in the cornea2. The excitation light from the light source 602 is directed through anaperture of a light source pinhole structure 604 a (solid line) oralternatively 604 b (dashed line), and then to a lens 610, such as anobjective lens or similar optical element. The lens 610 focuses theexcitation light to an area of the cornea 2 along the x-y plane at agiven depth along the z-axis.

The area of excited corneal tissue along the x-y plane is notperpendicular to the illumination path 600 a for the excitation light.In other words, the illumination path 600 a extends to the x-y plane atthe given depth at an angle that is not equal to 90°. As such, theillumination path 600 a is considered to be off-axis.

In response to the excitation light, the cross-linking agent in thecornea 2 fluoresces. The fluorescence emission travels through a lens614 and a color filter 612. The lens 614 focuses the fluorescenceemission onto a detector 618. The color filter 612 blocks residualexcitation light and/or other light not having a wavelength of thefluorescence emission. The fluorescence emission passes through anaperture of a detector pinhole structure 616 a (solid line) or 616 b(dashed line), which prevents light originating from above or below thegiven corneal depth from reaching the detector 618. The detector 618 maybe a photodiode, a photomultiplier tube, or a camera. The detector 618may be employed to quantify the amount, e.g., intensity, of fluorescenceemitted from the excited area of the cornea at the given depth.

The area of excited corneal tissue along the x-y plane is notperpendicular to the imaging path 600 b for the fluorescence emission.In other words, the imaging path 600 b extends from the x-y plane at thegiven depth at an angle that is not equal to 90°. As such, the imagingpath 600 b is considered to be off-axis.

According to one embodiment, the image plane of the detector 618 isparallel to the lens plane of the lens 614. As shown in FIG. 6, thedetector pinhole structure 616 a is also parallel to the image plane andthe lens plane and perpendicular to the imaging path 600 b.Correspondingly, the light source pinhole structure 604 a is alsoperpendicular to the illumination path 600 a.

According to an alternative embodiment, the image plane of the detector618 is not parallel to the lens plane of the lens 614. Instead, thedetector 618 and the lens 614 are arranged so that the image plane andthe lens plane are in a Scheimpflug configuration. In thisconfiguration, the excited area of corneal tissue along the x-y plane ismore uniformly in focus at the detector 618, even though the imagingpath 600 b is not perpendicular to the excited area of corneal tissue.The detector pinhole structure 616 b is angled relative to the imageplane and the lens plane and not perpendicular to the imaging path 600b. Correspondingly, the light source 602 and the lens 610 are arrangedso that the light source plane and the lens plane are in a Scheimpflugconfiguration. Additionally, the light source pinhole structure 604 b isangled relative to the light source plane and the lens plane and notperpendicular to the illumination path 600 a. In this configuration, theexcitation light is focused more uniformly to the excited area ofcorneal tissue along the x-y plane. The light source pinhole structure604 b and the detector pinhole structure 616 b may be parallel to thex-y plane.

The example systems described above may employ pinhole apertures tocreate a single point illumination; however, a pinhole array or slitillumination may be alternatively employed. In cases where an examplesystem is incorporated into a cross-linking treatment system asdescribed above, slit illumination can be generated via a DMD. Althoughslit illumination may be employed for illumination, a pinhole apertureor perpendicular slit in the imaging path may be sufficient to collectthe fluorescence emission.

In cases where the illumination path employs a Scheimpflugconfiguration, as shown for instance in FIG. 6, a dots array may begenerated using masks, gratings, acoustic optics, diffraction opticalelement (DOE), etc. The dots array is aligned along the z-axis and hasseparation larger than the axial resolution of the imaging path.

In the example systems 500, 600, the fluorescence emission is collectedfrom a smaller corneal region defined by an overlap between theillumination and imaging paths. As such, a smaller volume of cornealtissue can be interrogated by the systems 500, 600. Advantageously, thismay allow the optical requirements or constraints on the pinholestructures and numerical apertures of the respective lenses to berelaxed, which may in turn allow a larger working distance between thesystems 500, 600 and the subject.

Relative motion between the eye and the example system 300, 400, 500, or600, i.e., scanning, allows the system to generate and measurefluorescence emission at different depths along the z-axis. The examplesystems might scan only along the z-axis and not along the x-and y-axes.Scanning along the z-axis can be achieved according to variousapproaches. According to one approach, aspects of a system can beactively moved along the z-axis while the subject's eye remains at afixed position x, y, z. The entire system or certain components can bemoved along the z-axis by a motor or the like.

According to another approach, a system can scan along the z-axis byactively moving the subject's eye at a high frequency. In this approach,the subject's head may be situated in a chair that can be moved up anddown along the z-axis for a small range at high frequency while thesystem remains at a fixed position x, y, z.

To demonstrate further aspects of the present disclosure, FIG. 7illustrates an example system 700 for detecting fluorescence associatedwith a distribution of a cross-linking agent, e.g., riboflavin, in acornea 2. The system 700 includes an illumination path 700 a thatprojects a slit light pattern 10 into tissue, e.g., stroma, of thecornea 2 via a slit lamp. For instance, to generate the light pattern10, the system may employ a light source 702 that directs an excitationlight to at least a collector lens 704, a slit 706, and an objectivelens 708, which are configured according to the principles of Kohlerillumination. As shown in FIG. 7, the light source 702 may include afilament, and with the Kohler configuration, the filament is imaged onthe objective lens 708 while the slit 706 is imaged at the cornea 2.

The x-, y-, z-axes shown in FIG. 7 define an object space correspondingto the light pattern 10 received by the cornea 2. The light pattern 10extends from anterior to posterior along the x-z plane. Thecross-linking agent exposed to the light pattern 10 in the cornea 2 isexcited and emits fluorescence. The system 700 includes an imaging path700 b, which includes at least a Scheimpflug lens 710. The Scheimpfluglens 710 transmits the fluorescence emission to a detector 712. Amagnified image 10′ of the pattern 10 is created at the image plane ofthe detector 712. The x′-, y′-, z′-axes shown in FIG. 7 define an imagespace corresponding to the image 10′. A bandpass filter may be employedin the imaging path to attenuate the excitation light and other straylight.

In one embodiment, the system 700 employs a long movable slit aperture714 a in the image plane that can scan along the z′-axis. The aperture714 a is scanned over an array of pixels, e.g., a CCD array,corresponding to the fluorescence emission. Scanning the aperture alongthe z′-axis over a proper range yields the fluorescence intensityprofile in the cornea 2. This profile is recorded for the z-direction(direction of corneal depth) in the object space and is averaged in thex-direction over the slit length (mapped to the object space).

The example system 700 provides fast acquisition of the depth profilesdue to a relatively large aperture size. The profiles, however, areunresolved in the lateral direction, i.e., along the x-axis. If acertain spatial resolution in the lateral direction is desired,alternative embodiments may employ a lenticular array of pinholeapertures 714 b rather than the slit aperture 714 a. A single scan ofthe pinhole aperture array 714 b along the z′-axis yields atwo-dimensional profile of fluorescence intensity spatially resolved inboth the z-direction (direction of corneal depth) and the x-direction(lateral direction). The aperture size (width of slit aperture, pinholeaperture diameter) may be selected to balance acquisition rate andspatial resolution.

FIG. 8 illustrates aspects of another example system 800 that acquires athree-dimensional profile of the fluorescence emission due to theresponse to excitation light of a cross-linking agent in the cornea 2.The system 800 includes an illumination path 800 a for providing theexcitation light. The illumination path 800 a, for instance, may begenerated according to any of the embodiments described above. Tocapture the fluorescence emission, the system 800 includes a firstimaging path 800 b and a second imaging path 800 c. The system 800rotates the illumination path 800 a and the first and second imagingpaths 800 b, c around the eye by 180° to yield a dataset of images,which provide the three-dimensional profile. The three-dimensionalprofile indicates the presence of cross-linking agent in a volume ofcorneal tissue. The may involve several steps, e.g.:

-   -   1. Deconvolution—removing the blurring effect related to the        finite aperture size.    -   2. Correction for variable magnification of Scheimpflug lens        over the field of view.    -   3. Refraction correction—removing the image distortion due to        the refraction of light at the anterior corneal surface.    -   4. Smoothing—removing high spatial frequency components of the        image due to shot noise (small number of photons per pixel) and        diffractive patterns.    -   5. Corrections for eye movements during the recording time of        the image.        A proper calibration procedure may be developed for the image        distortion correction.

According to aspects of the present invention, embodiments may include atwo-channel design including illumination and detection channels, asshown for example in FIG. 7.

Embodiments may include an illumination channel providing a collimatedslit or pixel beam in the corneal stroma, as shown for example in FIG.7.

Embodiments may include an eye-tracking channel.

Embodiments may include an illumination channel that is a part of eyetracking optics (e.g., tracking of corneal apex with Purkinje images).

Embodiments may include an illumination channel that may include one ofthe following: a commercial or OEM slit lamp, or pixel projectionoptics.

Embodiments may include an illumination beam that acts as a source ofstray light propagating in different directions. Physical mechanisms ofthe stray light generation may include riboflavin fluorescence,cross-Link fluorescence and/or bulk scattering of stromal tissue.

Embodiments may include a detection channel that picks up the straylight transferring the light rays to image plane through a highnumerical aperture (NA) lens, as shown for example in FIG. 7. The objectplane of the lens (conjugate to the image plane) is aligned to theillumination beam in stroma using the Scheimpflug principle, as shownfor example in FIG. 7. Using the Scheimpflug principle makes it possibleto image the illuminated slit or other pattern between the anterior andposterior corneal surfaces at a convenient angle, e.g., 45°, as shownfor example in FIG. 7.

The thickness of the optical slice contributing to the image may besignificantly reduced by using high NA lens and confocal principal. Thelatter principle means that a small aperture is scanned over the imageplane while the image is recorded. In some embodiments, a CCD array maybe used where the pixels are switched on in certain order until thewhole area is covered. The pixel switching order must be chosen tominimize their cross-talk (either electrical or optical).

According to further aspects of the present disclosure, embodiments maybe employed to provide a high-resolution, non-contact pachymeter. Asdescribed above, embodiments can quantify the amount, e.g., intensity,of fluorescence emitted by a cross-linking agent in the cornea atvarious depths in response to an excitation light. For instance, asshown in FIG. 3, the system 300 can scan the cornea 2 at various depthsalong the z-axis to measure concentration of cross-linking agent as afunction of corneal depth. Such a scan may be sufficient to assess theentire thickness of the cornea. In particular, the scan may extend fromat least the interface between external air and the cornea (i.e.,anterior surface of the cornea) to the interface between the endotheliumand the anterior chamber (i.e., posterior surface of the cornea). Asalso described above, when a detector of an embodiment capturesfluorescence emission from the anterior surface of the cornea, there isa sudden increase in fluorescence signal followed by a gradual declinein the fluorescence signal as the scan moves deeper from the anteriorsurface into the stroma. When the scan reaches the interface between theendothelium and the anterior chamber (posterior surface), thefluorescence signal suddenly decreases to zero. By measuring thedistance between the depths at which the sudden increase (signal peak)and the sudden decrease (to zero) in fluorescence signal are detected,the corneal thickness can be determined. Thus, in addition to providinginformation on the presence of a cross-linking agent in the cornea,embodiments can provide a measurement of corneal thickness. Ameasurement of corneal thickness is typically required prior to across-linking treatment to ensure safety of the endothelium.

To reduce measurement noise and to obtain a more accurate measurement ofcorneal thickness, multiple scans extending through the thickness of thecornea (e.g., along the z-axis as shown in FIG. 3) may be conducted atdifferent transverse positions across the cornea (e.g., at different (x,y) positions as shown in FIG. 3). The measurements from these multiplescans can then be averaged. A pachymetry map covering a predefined oruser-selected set of transverse positions may be generated to assess,for instance, central corneal thickness and/or peripheral cornealthickness. For instance, a pachymetry map may be generated over asection associated with a keratoconic defect.

Using aspects of confocal fluorescence microscopy, embodiments canachieve high resolution (e.g., less than 100 μm) and rapid (e.g., lessthan one second) measurement of corneal thickness. Additionally, suchmeasurement can be taken without physically contacting the cornea,thereby enhancing safety and comfort for the subject. Furthermore, thisapproach to pachymetry is insensitive to variations in density,hydration state, and refractive properties of the cornea. Also, thisapproach is insensitive to the presence of haze sometimes associatedwith eyes, especially with diseased eyes. As such, this approach canprovide more accurate measurements of corneal thickness in contrast, forinstance, with ultrasound-based approaches, which rely on a singlenominal speed of sound to measure corneal thickness.

Rather than detecting signals based on fluorescence emission from across-linking agent in a cornea in response to excitation light,alternative implementations of the embodiments above can determinecorneal thickness by detecting signals based on incidence light that isreflected from a cornea, where no cross-linking agent is present in thecornea. The reflected light from a scan through the cornea providessignals that indicate the presence of the anterior surface of the corneaand the posterior surface of the cornea. In particular, signal spikes orother signal changes may mark the anterior and posterior surfaces of thecornea. Advantageously, this alternative implementation can providereliable measurements of corneal thickness independent of any presenceof cross-linking agent in the cornea.

In summary, FIG. 9 generally illustrates an example approach 900 formeasuring corneal thickness via embodiments that employ aspects ofconfocal microscopy. In act 902, for one or more positions (x, y) acrossa cornea, light is directed to the cornea through various corneal depths(z) via a configured confocal microscope system. In act 904,fluorescence emission from a cross-linking agent in the cornea orreflected light from the cornea without a cross-linking agent isdetected from the various corneal depths. In act 906, a first signal anda corresponding first depth associated with the anterior surface of thecornea is detected from the fluorescence emission/reflected light. Inact 908, a second signal and a corresponding second depth associatedwith the posterior surface of the cornea is detected from thefluorescence emission/reflected light. In act 910, a corneal thicknessis determined from a distance between the first depth and the seconddepth. In optional act 912, if there is more than one position (x, y)across the cornea, the corneal thicknesses from the more than onepositions are averaged to determine a more accurate measurement.

As described above, according to some aspects of the present disclosure,some or all of the steps of the above-described and illustratedprocedures can be automated or guided under the control of a controller(e.g., the controller 120). Generally, the controllers may beimplemented as a combination of hardware and software elements. Thehardware aspects may include combinations of operatively coupledhardware components including microprocessors, logical circuitry,communication/networking ports, digital filters, memory, or logicalcircuitry. The controller may be adapted to perform operations specifiedby a computer-executable code, which may be stored on a computerreadable medium.

As described above, the controller may be a programmable processingdevice, such as an external conventional computer or an on-board fieldprogrammable gate array (FPGA) or digital signal processor (DSP), thatexecutes software, or stored instructions. In general, physicalprocessors and/or machines employed by embodiments of the presentdisclosure for any processing or evaluation may include one or morenetworked or non-networked general purpose computer systems,microprocessors, field programmable gate arrays (FPGA's), digital signalprocessors (DSP's), micro-controllers, and the like, programmedaccording to the teachings of the example embodiments of the presentdisclosure, as is appreciated by those skilled in the computer andsoftware arts. The physical processors and/or machines may be externallynetworked with the image capture device(s), or may be integrated toreside within the image capture device. Appropriate software can bereadily prepared by programmers of ordinary skill based on the teachingsof the example embodiments, as is appreciated by those skilled in thesoftware art. In addition, the devices and subsystems of the exampleembodiments can be implemented by the preparation ofapplication-specific integrated circuits or by interconnecting anappropriate network of conventional component circuits, as isappreciated by those skilled in the electrical art(s). Thus, the exampleembodiments are not limited to any specific combination of hardwarecircuitry and/or software.

Stored on any one or on a combination of computer readable media, theexample embodiments of the present disclosure may include software forcontrolling the devices and subsystems of the example embodiments, fordriving the devices and subsystems of the example embodiments, forenabling the devices and subsystems of the example embodiments tointeract with a human user, and the like. Such software can include, butis not limited to, device drivers, firmware, operating systems,development tools, applications software, and the like. Such computerreadable media further can include the computer program product of anembodiment of the present disclosure for performing all or a portion (ifprocessing is distributed) of the processing performed inimplementations. Computer code devices of the example embodiments of thepresent disclosure can include any suitable interpretable or executablecode mechanism, including but not limited to scripts, interpretableprograms, dynamic link libraries (DLLs), Java classes and applets,complete executable programs, and the like. Moreover, parts of theprocessing of the example embodiments of the present disclosure can bedistributed for better performance, reliability, cost, and the like.

Common forms of computer-readable media may include, for example, afloppy disk, a flexible disk, hard disk, magnetic tape, any othersuitable magnetic medium, a CD-ROM, CDRW, DVD, any other suitableoptical medium, punch cards, paper tape, optical mark sheets, any othersuitable physical medium with patterns of holes or other opticallyrecognizable indicia, a RAM, a PROM, an EPROM, a FLASH-EPROM, any othersuitable memory chip or cartridge, a carrier wave or any other suitablemedium from which a computer can read.

While the present disclosure has been described with reference to one ormore particular embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present disclosure. Each of these embodiments andobvious variations thereof is contemplated as falling within the spiritand scope of the present disclosure. It is also contemplated thatadditional embodiments according to aspects of the present disclosuremay combine any number of features from any of the embodiments describedherein.

What is claimed is:
 1. A measurement system for a cornea, comprising: alight source configured to emit an excitation light that causes afluorescing agent applied to a cornea to generate a fluorescenceemission; at least one optical element positioned to receive theexcitation light from the light source and configured to focus theexcitation light to an area of corneal tissue at a selected depth of thecornea, the fluorescing agent in the cornea generating the fluorescenceemission in response to the excitation light; a pinhole structureincluding an aperture, the pinhole structure positioned to receive thefluorescence emission from the fluorescing agent in the cornea, theaperture being configured to selectively transmit the fluorescenceemission from the area of corneal tissue at the selected depth; adetector positioned to capture the selected fluorescence emissiontransmitted by the aperture and configured to communicate informationrelating to a measurement of the selected fluorescence emission capturedby the detector; and a controller communicatively coupled to thedetector and configured to receive the information from the detector andto determine, based on the information, a measurement of the fluorescingagent in the area of corneal tissue at the selected depth.
 2. Themeasurement system of claim 1, wherein the measurement of thefluorescing agent is determined according to a resolution of greaterthan 10 μm along an axis corresponding to the depth of the cornealtissue and/or a plane transverse to the axis.
 3. The measurement systemof claim 1, further comprising a scan mechanism configured to cause theat least one optical element to scan the cornea at a plurality of depthsand to focus the excitation light on a respective area of corneal tissueat each depth, wherein for each depth: (i) the aperture of the pinholestructure is configured to selectively transmit the fluorescenceemission from the respective area of corneal tissue, and (ii) thedetector is configured to capture the selected fluorescence emissiontransmitted by the aperture and to communicate information relating to ameasurement of the selected fluorescence emission captured by thedetector, and the controller is configured to receive the informationfrom the detector for each depth and to determine, based on theinformation for the plurality of depths, a measurement of thefluorescing agent in the cornea as a function of depth.
 4. Themeasurement system of claim 2, wherein the plurality of depths extendfrom an anterior surface of the cornea to a posterior surface of thecornea, and the controller is further configured to determine, based onthe information for the plurality of depths, at least one of a locationof the posterior surface, a distance between the anterior surface andthe posterior surface, or a location of an interface between sections ofthe cornea.
 5. The measurement system of claim 3, wherein to determineat least one of the location of the posterior surface, the distancebetween the anterior surface and the posterior surface, or the locationof an interface between sections of the cornea, the controller evaluateschanges in the measurements of the fluorescing agent in the corneaacross the plurality of depths, the changes corresponding to structuralcharacteristics of the cornea.
 6. The measurement system of claim 2,wherein the controller is further configured to detect an incompletescan of the cornea by evaluating changes in the measurements of thefluorescing agent in the cornea across the plurality of depths, thechanges corresponding to structural characteristics of the cornea. 7.The measurement system of claim 2, wherein the scan mechanism isconfigured to cause the at least one optical element to scan, more thanone time, the cornea at the plurality of depths, and the controller isfurther configured to determine, based on the information from the morethan one scans, an amount of the fluorescing agent in the cornea as afunction of depth.
 8. The measurement system of claim 2, wherein theplurality of depths extend from an anterior surface of the cornea to atleast approximately 200 μm into the cornea, and adjacent depths in theplurality of depths are separated by approximately 10 μm toapproximately 100 μm.
 9. The measurement system of claim 2, wherein thescan mechanism is further configured to operate the at least one opticalelement to scan the cornea at a plurality of transverse locations acrosseach depth and to focus the excitation light on a respective area ofcorneal tissue at each transverse location at each depth, wherein foreach transverse location at each depth: (i) the aperture of the pinholestructure is configured to selectively transmit the fluorescenceemission from the respective area of corneal tissue, and (ii) thedetector is configured to capture the selected fluorescence emissiontransmitted by the aperture and to communicate information relating to ameasurement of the selected fluorescence emission captured by thedetector, and the controller is configured to receive the informationfrom the detector for each transverse location and to determine, basedon the information for the plurality of the transverse locations at theplurality of depths, a measurement of the fluorescing agent in thecornea as a function of transverse location and depth.
 10. Themeasurement system of claim 8, wherein the plurality of depths extendfrom an anterior surface of the cornea to a posterior surface of thecornea, and the controller is further configured to determine, based onthe information for the plurality of the transverse locations at theplurality of depths, at least one of a location of the posteriorsurface, a distance between the anterior surface and the posteriorsurface, or a location of an interface between sections of the cornea.11. The measurement system of claim 1, wherein the fluorescing agent isa cross-linking agent, and the controller is further configured tocommunicate that the measurement of the cross-linking agent in thecornea at the selected depth of the cornea meets a threshold forstarting a cross-linking treatment for the cornea.
 12. A measurementsystem for a cornea, comprising: a light source configured to emit anincidence light; at least one optical element positioned to receive theincidence light from the light source and configured to focus theincidence light to an area of corneal tissue at a selected depth of thecornea, the area of corneal tissue reflecting the incidence light; ascan mechanism configured to cause the at least one optical element toscan the cornea at a plurality of depths and to focus the incidencelight on a respective area of corneal tissue at each depth, theplurality of depths extending from an anterior surface of the cornea toa posterior surface of the cornea; a pinhole structure including anaperture, the pinhole structure positioned to receive, for each depth,the reflected light from the respective area of corneal tissue, theaperture of the pinhole structure configured, for each depth, toselectively transmit the reflected light from the respective area ofcorneal tissue; a detector positioned to capture the selected reflectedlight transmitted by the aperture for each depth and configured tocommunicate, for each depth, information relating to a measurement ofthe selected reflected light captured by the detector; and a controllercommunicatively coupled to the detector and configured to receive theinformation from the detector for each depth and to determine, based onthe information for the plurality of depths, at least one of a locationof the posterior surface, a distance between the anterior surface andthe posterior surface, or a location of a sub-corneal interface.
 13. Themeasurement system of claim 12, wherein the measurement of the selectedreflected light is determined according to a resolution of greater than10 μm along an axis corresponding to the depth of the corneal tissueand/or a plane transverse to the axis.
 14. The measurement system ofclaim 12, wherein to determine at least one of a location of theposterior surface, the distance between the anterior surface and theposterior surface, or the location of an interface between sections ofthe cornea, the controller evaluates changes in the measurements of thereflected light across the plurality of depths, the changescorresponding to structural characteristics of the cornea.
 15. Themeasurement system of claim 12, wherein the controller is furtherconfigured to detect an incomplete scan of the cornea by evaluatingchanges in the measurements of the reflected light in the cornea acrossthe plurality of depths, the changes corresponding to structuralcharacteristics of the cornea.
 16. The measurement system of claim 12,wherein the scan mechanism is configured to cause the measurement systemto scan, more than one time, the cornea at a plurality of depths, andthe controller is further configured to determine, based on theinformation from the more than one scans, at least one of the locationof the posterior surface, the distance between the anterior surface andthe posterior surface, or the location of a sub-corneal interface. 17.The measurement system of claim 12, wherein the scan mechanism isfurther configured to cause the at least one optical element to scan thecornea at a plurality of transverse locations along each depth and tofocus the incidence light on a respective area of corneal tissue at eachtransverse location at each depth, wherein for each transverse locationalong each depth: (i) the aperture of the pinhole structure isconfigured to selectively transmit the reflected light from therespective area of corneal tissue, and (ii) the detector is configuredto capture the selected reflected light transmitted by the aperture andconfigured to communicate information relating to a measurement of theselected reflected light captured by the detector, and the controller isconfigured to receive the information from the detector for eachtransverse location and to determine, based on the information forplurality of the transverse locations at the plurality of depths, atleast one of the location of the posterior surface, the distance betweenthe anterior surface and the posterior surface, or the location of asub-corneal interface.
 18. A measurement system for a cornea,comprising: a light source configured to emit an excitation light thatcauses a fluorescing agent applied to a cornea to generate afluorescence emission; at least one optical element positioned toreceive the excitation light from the light source and configured todeliver the excitation light, the excitation light extending through aplurality of depths of the cornea, the fluorescing agent in the corneagenerating the fluorescence emission in response to the excitationlight; a detector positioned to capture an image of the fluorescenceemission from the cornea; and a controller communicatively coupled tothe detector and configured to: receive the image from the detector,scan the image to measure the fluorescence emission at the plurality ofdepths, and to determine a measurement of the fluorescing agent in thecornea as a function of depth based on the measurement of thefluorescence emission at the plurality of depths.
 19. The measurementsystem of claim 18, wherein the controller scans the image with a slitaperture that scans an array of pixels along the plurality of depths.20. The measurement system of claim 18, wherein the excitation lightfurther extends in at least one lateral direction as it extends throughthe plurality of depths of the cornea, and the controller is furtherconfigured to scan the image to measure the fluorescence emission alongthe at least one lateral direction at the plurality of depths, and todetermine a measurement of the fluorescing agent in the cornea as afunction of depth and lateral location based on the measurement of thefluorescence emission along the at least one transverse direction at theplurality of depths, and the controller scans the image with alenticular array of pinhole apertures that scans an array of lateralpixels along the plurality of depths through the cornea.